Protein phosphorylation is widely accepted as one of the principle mechanisms in the control of almost all cellular processes. Studies over the last 25 years have provided evidence that protein phosphorylation plays a major role in the regulation of neuronal function. Protein tyrosine kinases, a unique class of protein kinases that exclusively phosphorylate tyrosine residues of their substrate proteins, have recently been shown to be abundant in neurons. However, the role of protein tyrosine kinases in the regulation of neuronal function is not clear. The nicotinic acetylcholine receptor (AChR) is the neurotransmitter-gated ion channel which mediates synaptic transmission at the neuromuscular junction (NMJ) and is the most well-characterized neurotransmitter receptor in biology. Studies in our laboratory have demonstrated that the AChR is phosphorylated on tyrosine residues in vitro and in vivo. We have previously identified two protein tyrosine kinases, fyn and fyk, which phosphorylate the AChR and have characterized the major protein tyrosine phosphatase that dephosphorylates the AChR. In addition, studies in our laboratory have shown that tyrosine phosphorylation of the AChR is regulated by the postsynaptic neuron via a nerve-derived extracellular matrix protein, agrin, that mediates nerve-induced clustering of the AChR at synaptic contacts. Moreover, in recent experiments we have found that tyrosine phosphorylation of the AChR is regulated by a postsynaptic 43 kDa protein that regulates AChR clustering. These results suggest that tyrosine phosphorylation may be important in nerve-, agrin- and 43 kDa- induced clustering of the AChR. The specific aim of this research proposal is to continue to characterize the role of tyrosine phosphorylation in the regulation of the function of the AChR and the NMJ. To accomplish these goals we will examine the regulation of the fyn and fyk protein tyrosine kinases by agrin and the 43 kDa protein. Proteins that directly interact with the fyn and fyk protein tyrosine kinases, the 43 kDa protein and other synaptic proteins will be identified by coimmunoprecipitation, fusion protein affinity chromatography and the yeast two-hybrid system. In addition, synaptic phosphotyrosine-containing proteins involved in AChR clustering will be cloned using expression cloning techniques. These studies will define the signal transduction pathways for the regulation of tyrosine phosphorylation of the AChR and other synaptic proteins. They will also identify cytoskeletal proteins involved in AChR clustering and the role of tyrosine phosphorylation of these proteins in the regulation of their function. The proposed research will provide a better understanding of the basic regulatory mechanisms involved in the modulation of synaptic transmission at the NMJ. Such knowledge is essential for understanding the function of the nervous system in health and in disease.